1. Field of the Invention
The present invention relates to the use of cyclic dinucleotides, such as c-di-GMP and its analogues, to inhibit and treat cancer. The cyclic dinucleotides in particular inhibit basal and growth factor-induced human cancer cell proliferation.
2. Description of the Related Art
Experimental data indicate that most colon cancers arise as a consequence of progression from normal colonic mucosa to adenomatous polyp to cancer, associated with the accumulation of somatic genetic alterations that affect the regulation of apoptosis and DNA repair (Kinzler et al., 1996; and Jass et al., 2002). These alterations include mutations and methylation of oncogenes, tumor suppressor and mismatch repair genes (Kinzler et al., 1996; and Jass et al., 2002). Environmental factors (e.g., fecal bile acid concentrations) play an important promoting role in this process (Hill et al., 1975; Reddy et al., 1977; Hill, 1991; and Lichtenstein et al., 2000).
The American Cancer Society estimates that 106,370 cases of colon cancer will be diagnosed in 2004 (cancer.org). In the United States, colon cancer is the second most frequent cause of cancer death and the leading gastrointestinal cause of death (Russo et al., 2004). The outcome is strongly correlated with cancer stage at the time of diagnosis (Bresalier, 2002). Patients with Stage 0 disease (limited to the mucosa) have a more than 90% survival (Bresalier, 2002). In contrast, those with Stage III disease that has spread outside the colon to one or more lymph nodes have a 5-year survival approximating only 55% (Bresalier, 2002). Adjuvant chemotherapy for patients with Stage II or III disease with 5-fluorouracil (5-FU) plus levamisole or leucovorin may reduce colon cancer recurrence and mortality by 42 and 33%, respectively (Bresalier, 2002). Although the use of 5-FU in combination with topoisomerase inhibitors, such as irinotecan, thymidylate synthase inhibitors, and other agents shows promise, treatment for advanced disease (Stages III and IV) remains only marginally effective (Bresalier, 2002). Recently, based on efficacy in clinical trials, a synthetic chimeric monoclonal antibody, cetuximab (Erbitux), that is specific for EGFR was FDA-approved, in combination with irinotecan, for treatment of metastatic colorectal cancer in patients who are refractory to irinotecan-based chemotherapy. Cetuximab is also approved as a single agent to treat patients with EGFR-expressing, metastatic colorectal cancer who are intolerant to irinotecan-based chemotherapy.
The development of cancer requires that the cell overcome normal restrictions placed on cell growth. Normal cell growth is ultimately regulated by the cell cycle. The cell cycle comprises the events that occur in the nucleus between two cell divisions. The cycle is divided into 4 phases called G1 (first gap), S (DNA synthesis), G2 (second gap), and M (mitosis). The RNA and proteins needed for DNA replication are synthesized during the G1 phase. In S phase, DNA replication takes place and the cell's DNA content doubles from the diploid value of 2n to the fully replicated, tetraploid value of 4n. The tetraploid cell prepares for the upcoming mitotic division in G2 phase. Finally, in M phase the cell divides into two daughter cells, each containing a diploid (2n) complement of DNA. Cells may also withdraw from the cell cycle to enter a quiescent state termed G0. Under certain conditions, cells can be stimulated to leave the G0 phase and reenter the cell cycle. The major regulatory point controlling entry from G1 into S phase of the cell cycle occurs late in G1 and is termed the restriction (R) point. Growth of cancer cells may by stimulated by a number of growth factors, including epidermal growth factor (EGF) and acetylcholine, that interact with specific growth factor receptors on cancer cells.
Post-receptor signaling cascades are crucial for ligand-receptor interaction to result in changes in cell function. The muscarinic cholinergic family of G protein-coupled receptors (GPCRs) includes five muscarinic receptor subtypes desgignated M1-M5 (Bonner et al., 1987; and Brann et al., 1993). Peripheral M3 receptors, are very common in the gastrointestinal tract are coupled to a G protein. It is apparent that some cancer cells express M3 muscarinic receptors and activation of these receptors results in stimulation of cancer cell proliferation; however, the cellular pathways underlying these events have not been elucidated. In contrast to GPCRs, epidermal growth factor receptor (EGFR) is a member of the growth factor receptor family and activation of these receptors leads to proliferation of cancer cells (Murphy et al., 2001). EGFR is commonly overexpressed in a number of epithelial malignancies and is often associated with an aggressive phenotype and EGFR is present in over 50% of cases of lung cancer, head and neck squamous cell carcinomas and colon cancer (Janmaat et al., 2003). Recently, it has been found that activation of GPCRs may result in transctivation of receptors such as EGFR (Zhang et al., 1999).
Cyclic nucleotides, such as cAMP and cGMP, are well recognized as important low-molecular weight signaling molecules in eukaryotes. In bacteria, while cAMP has a role in alleviating glucose catabolite repression (Jackson et al., 2002; Notley-McRobb et al., 1997), cGMP has not been shown to act as a signaling molecule. However, another guanosine nucleotide, the cyclic dinucleotide c-di-GMP (also known as 3′,5′-cyclic diguanylic acid, cyclic diguanylate, cyclic diguanosine monophosphate, cyclic bis (3′→15′) diguanylic acid, cyclic diguanylic acid, cGpGp, and c-GpGp)

where G in the above structure is guanine, has been reported to be an intracellular bacterial signaling molecule in a few species and whose structure is known and consists of two cGMP molecules bound head-to-tail (Jenal, 2004 and Ross et al., 1991). c-di-GMP was first identified in Acetobacter xylinum (renamed Gluconacetobacter xylinum) and shown to regulate cellulose production in this species (Amikam et al., 1989; Mayer et al., 1991; Ross et al., 1990 and 1991). The exact molecular mechanism remains unclear but regulation in G. xylinum appears to involve c-di-GMP binding to a membrane protein that activates gene expression. Cellulose production appears to be modulated by the opposing effects of two proteins with GGDEF domains, diguanylate cyclase (Dgc) and c-di-GMP phosphodiesterase (PdeA), each controlling the level of c-di-GMP in the cell. Thus, c-di-GMP is thought to be a signaling molecule. Proteins containing GGDEF domains are very widespread in microbial cells suggesting that they are naturally produced by many microorganisms, while they appear to be much less prevalent in human proteins.
The use of unmethylated oligonucleotides in the treatment or prevention of cancer has been previously reported. Synthetic oligonucleotides containing CpG with appropriate flanking regions (CpG motif) have been found to activate macrophages, dendritic cells and B cells to secrete a variety of immunomodulatory cytokines such as IL-6, IL-12, IL-18 and gamma interferon (Krieg, 2002). CpG DNA has also been shown to activate costimulatory molecules such as CD80 and CD86 and to induce strong innate immunity at mucosal surfaces. The immunostimulatory property of CpG DNA produces long-term vaccine-like effects due to its adjuvant properties. CpG oligonucleotides influence both antibody and cell-mediated immunity and applications include vaccine adjuvants, taming allergic reactions and potentiating monoclonal antibodies and cytotoxic immune cells. They also enhance the antitumor effects of chemotherapeutic agents and improve survival after surgical section of a solid tumor (Weigel et al., 2003). For CpG oligonucleotides, the anti-tumor effect is mediated via activation of the host immune system, not through direct anti-tumor effects. Based on their immunotherapeutic properties, CpG oligonucleotides have been used to treat and prevent various cancers and used in cancer vaccines (U.S. Pat. Nos. 6,653,292; 6,429,199; 6,406,705; and 6,194,388). These CpG oligonucleotides however are not cyclic.
Cyclic dinucleotides are reported to cause cell cycle arrest (Steinberger et al., 1999). However, their application to the treatment of cancer has not been proposed or studied. Approximately 60,000 Americans die from colon cancer each year (Steinberger et al., 1999; and Winawer et al., 2003). Although surgical treatment is effective for early lesions, treatment for advanced metastatic disease is not very effective with very low survival rates for patients with invasive and metastatic colon cancer. Hence, a safe therapeutic (anti-cancer) agent that inhibits colon cancer progression, and cause cancer involution and regression would be extremely beneficial.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.